The potential of extracts of Caryocar villosum pulp to scavenge reactive oxygen and nitrogen species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Caryocar villosum (piquid) is a native fruit from the Amazonian region, considered to be an interesting source of bioactive compounds. In this paper, five extracts of C. villosum pulp were obtained, using solvents with different polarities and their in vitro scavenging capacity against reactive oxygen species (ROS) and reactive nitrogen species (RNS) was determined. Additionally, the phenolic compounds and carotenoids in each extract were identified and quantified by a high performance liquid chromatography coupled to diode array and mass spectrometer detectors (HPLC-DAD-MS/MS). The ethanol/water and water extracts, which presented the highest phenolic contents (5163 and 1745 mu g/g extract, respectively), with ellagic acid as the major phenolic compound, proved to have the highest ROS and RNS scavenging potential. Nevertheless, in general, ellagic acid was less effective in scavenging ROS (IC50 from 1.7 to 108 mu g/ml) and RNS (IC50 from 0.05 to 0.59 mu g/ml), when compared to gallic acid (IC50 from 0.4 to 226 mu g/ml for ROS and IC50 from 0.04 to 0.12 mu g/ml for RNS). The results obtained in the present study clearly demonstrated that the in vitro antioxidant efficiency of C. villosum extracts was closely related to their contents of phenolic compounds. (C) 2012 Elsevier Ltd. All rights reserved.135317401749Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Carotenoids inhibit lipid peroxidation and hemoglobin oxidation, but not the depletion of glutathione induced by ROS in human erythrocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Aims: Despite the presence of endogenous antioxidants in erythrocytes, these cells are highly susceptible to oxidative damage and some exogenous antioxidants, such as carotenoids, are able to inhibit the pro-oxidant effect provided by reactive oxygen species. In this study, we evaluated the potential of carotenoids usually detected in human blood plasma (beta-carotene, zeaxanthin, lutein, beta-cryptoxanthin and lycopene) to prevent the oxidative damage in erythrocytes. Main methods: Human erythrocytes were subjected to induced oxidative damage and the following biomarkers of oxidative stress were monitored: lipid peroxidation [induced by tert-butyl hydroperoxide (tBHP) or by 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH)] and AAPH-induced oxidation of hemoglobin and depletion of glutathione. Key findings: When tBHP was used to induce lipid peroxidation, lycopene was the most efficient carotenoid (IC50 = 2.2 +/- 0.4 mu M), while lutein was the most efficient (IC50 = 2.5 +/- 0.7 mu M) when peroxyl radicals (ROO center dot) were generated by AAPH. In relation to the hemoglobin oxidation induced by AAPH, beta-carotene and zeaxanthin were the most efficient antioxidants (IC50 = 2.9 +/- 0.3 mu M and 2.9 +/- 0.1 mu M, respectivelY). Surprisingly beta-cryptoxanthin and lycopene did not inhibit hemoglobin oxidation or lipid peroxidation when induced by AAPH, even at the highest tested concentration (3 mu M). Additionally, the tested carotenolds did not prevent ROO center dot-mediated GSH depletion and GSSG formation probably due to the lack of interaction between carotenoids (apolar) and glutathione (polar). Significance: Our study contributes with important insights that carotenoids may exert therapeutical potential to act as a natural antioxidant to prevent ROO center dot-induced toxicity in human erythrocytes. (C) 2014 Elsevier Inc. All rights reserved.99416715260Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao para a Ciencia e Tecnologia [SFRH/BPD/76909/2011]FSEMCTESFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [Proc. 2012/01356-0, 2013/07914-8]Fundacao para a Ciencia e Tecnologia [SFRH/BPD/76909/2011

Carotenoids Inhibit Lipid Peroxidation And Hemoglobin Oxidation, But Not The Depletion Of Glutathione Induced By Ros In Human Erythrocytes

Aims Despite the presence of endogenous antioxidants in erythrocytes, these cells are highly susceptible to oxidative damage and some exogenous antioxidants, such as carotenoids, are able to inhibit the pro-oxidant effect provided by reactive oxygen species. In this study, we evaluated the potential of carotenoids usually detected in human blood plasma (β-carotene, zeaxanthin, lutein, β-cryptoxanthin and lycopene) to prevent the oxidative damage in erythrocytes. Main methods Human erythrocytes were subjected to induced oxidative damage and the following biomarkers of oxidative stress were monitored: lipid peroxidation [induced by tert-butyl hydroperoxide (tBHP) or by 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH)] and AAPH-induced oxidation of hemoglobin and depletion of glutathione. Key findings When tBHP was used to induce lipid peroxidation, lycopene was the most efficient carotenoid (IC50 = 2.2 ± 0.4 μM), while lutein was the most efficient (IC50 = 2.5 ± 0.7 μM) when peroxyl radicals (ROO) were generated by AAPH. In relation to the hemoglobin oxidation induced by AAPH, β-carotene and zeaxanthin were the most efficient antioxidants (IC50 = 2.9 ± 0.3 μM and 2.9 ± 0.1 μM, respectively). Surprisingly β-cryptoxanthin and lycopene did not inhibit hemoglobin oxidation or lipid peroxidation when induced by AAPH, even at the highest tested concentration (3 μM). Additionally, the tested carotenoids did not prevent ROO-mediated GSH depletion and GSSG formation probably due to the lack of interaction between carotenoids (apolar) and glutathione (polar). Significance Our study contributes with important insights that carotenoids may exert therapeutical potential to act as a natural antioxidant to prevent ROO-induced toxicity in human erythrocytes. © 2014 Elsevier Inc.9901/02/155260Aksoy, Y., Öǧüş, I.H., Özer, N., The effect of tert-butylhydroperoxide on the thiol redox status in human erythrocytes and the protective role of glucose and antioxidants (2003) Turk J Chem, 27, pp. 433-443Barbosa, D.J., Capela, J.P., Oliveira, J.M.A., Silva, R., Ferreira, L.M., Pro-oxidant effects of ecstasy and its metabolites in mouse brain synaptosomes (2012) Br J Pharmacol, 165, pp. 1017-1033Berliner, J.A., Heinecke, J.W., The role of oxidized lipoproteins in atherogenesis (1996) Free Radic Biol Med, 20, pp. 707-727Claro, L.M., Leonart, M.S.S., Comar, S.R., Nascimento, A.J., Effect of vitamins C and e on oxidative processes in human erythrocytes (2006) Cell Biochem Funct, 24, pp. 531-535Di Mascio, P., Kaiser, S., Sies, H., Lycopene as the most efficient biological carotenoid singlet oxygen quencher (1989) Arch Biochem Biophys, 274, pp. 532-538Dwight, J.F.St.J., Hendry, B.M., The effects of tert-butyl hydroperoxide on human erythrocyte membrane ion transport and the protective actions of antioxidants (1996) Clin Chim Acta, 249, pp. 167-181El-Agamey, A., Lowe, G.M., McGarvey, D.J., Mortensen, A., Phillip, D.M., Carotenoid radical chemistry and antioxidant/pro-oxidant properties (2004) Arch Biochem Biophys, 430, pp. 37-48Ferrari, C.K.B., Lipid oxidation in food and biological systems: General mechanisms and nutritional and pathological implications (1998) Rev Nutr, 11, pp. 3-14Fujino, T., Watanabe, K., Beppu, M., Kikugawa, K., Yasuda, H., Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase (2000) Biochim Biophys Acta, 1478, pp. 102-112Gilbert, H.S., Stump, D.D., Roth, Jr.E.F., A method to correct for errors caused by generation of interfering compounds during erythrocyte lipid peroxidation (1984) Anal Biochem, 137, pp. 282-286Grinberg, L.N., Newmark, H., Kitrossky, N., Rahamim, E., Chevion, M., Protective effects of tea polyphenols against oxidative damage to red blood cells (1997) Biochem Pharmacol, 54, pp. 973-978Guo, J.J., Hu, C.H., Mechanism of chain termination in lipid peroxidation by carotenes: A theoretical study (2010) J Phys Chem B, 114, pp. 16948-16958Halliwell, B., Gutteridge, J.M.C., (2007) Free Radicals in Biology and Medicine, , Oxford University Press OxfordHammad, L.A., Wu, G., Saleh, M.M., Klouckova, I., Dobrolecki, L.E., Elevated levels of hydroxylated phosphocholine lipids in the blood serum of breast cancer patients (2009) Rapid Commun Mass Spectrom, 23, pp. 863-876Hininger, I.A., Meyer-Wenger, A., Moser, U., Wright, A., Southon, S., No significant effects of lutein, lycopene or β-carotene supplementation on biological markers of oxidative stress and LDL oxidizability in healthy adult subjects (2001) J Am Coll Nutr, 20, pp. 232-238Hseu, Y.C., Chang, W.C., Hseu, Y.T., Lee, C.H., Yech, Y.J., Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes (2002) Life Sci, 71, pp. 469-482Jomová, K., Kysel, O., Madden, J.C., Morris, H., Enoch, S.J., Electron transfer from all-trans-b-carotene to the t-butyl peroxyl radical at low oxygen pressure (an EPR spectroscopy and computational study) (2009) Chem Phys Lett, 478, pp. 266-270Kanias, T., Wong, K., Acker, J.P., Determination of lipid peroxidation in desiccated red blood cells (2007) Cell Preserv Technol, 5, pp. 165-174Kiko, T., Nakagawa, K., Tsuduki, T., Suzuki, T., Arai, H., Significance of lutein in red blood cells of Alzheimer's disease patients (2012) J Alzheimers Dis, 28, pp. 593-600Ko, F.N., Hsiao, G., Kuo, Y.H., Protection of oxidative hemolysis bydemethyldiisoeugenol in normal and β-thalassemicred blood cells (1997) Free Radic Biol Med, 22, pp. 215-222Kok, T.M.C.M., Waard, P., Wilms, L.C., Van Breda, S.G.J., Antioxidative and antigenotoxic properties of vegetables and dietary phytochemicals: The value of genomics biomarkers in molecular epidemiology (2010) Mol Nutr Food Res, 54, pp. 208-217Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., Protein measurement with the folin phenol reagent (1951) J Biol Chem, 193, pp. 265-275Magalhães, A.S., Silva, B.M., Pereira, J.A., Andrade, P.B., Valentão, P., Protective effect of quince (Cydonia oblonga Miller) fruit against oxidative hemolysis of human erythrocytes (2009) Food Chem Toxicol, 47, pp. 1372-1377Mercadante, A.Z., (2007) Carotenoids in Foods: Sources and Stability during Processing and Storage, pp. 213-235. , C. Socaciu, CRC Press New YorkMiyazawa, T., Suzuki, T., Yasuda, K., Fujimoto, K., Meguro, K., (1992) Accumulation of Phospholipid Hydroperoxides in Red Blood Cell Membranes in Alzheimer Disease, pp. 327-330. , K. Yagi, M. Kondo, E. Niki, T. Yoshikawa, Elsevier Science Publishing AmsterdamMohanty, J.G., Eckley, D.M., Williamson, J.D., Launer, L.J., Rifkind, J.M., Do red blood cell-β-amyloid interactions alter oxygen delivery in Alzheimer's disease? (2008) Adv Exp Med Biol, 614, pp. 29-35Müller, L., Fröhlich, K., Böhm, V., Comparative antioxidant activities of carotenoids measured by ferric reducing antioxidant power (FRAP), ABTS bleaching assay (αTEAC), DPPH assay and peroxyl radical scavenging assay (2011) Food Chem, 129, pp. 139-148Nagababu, E., Rifkind, J.M., Reaction of hydrogen peroxide with ferrylhemoglobin: Superoxide production and heme degradation (2000) Biochemistry, 39, pp. 12503-12511Nakagawa, K., Fujimoto, K., Miyazawa, T., β-Carotene as a high-potency antioxidant to prevent the formation of phospholipid hydroperoxides in red blood cells of mice (1996) Biochim Biophys Acta, 1299, pp. 110-116Nakagawa, K., Kiko, T., Hatade, K., Sookwong, P., Arai, H., Miyazawa, T., Antioxidant effect of lutein towards phospholipid hydroperoxidation in human erythrocytes (2009) Br J Nutr, 102, pp. 1280-1284Nakagawa, K., Kiko, T., Miyazawa, T., Burdeos, G.C., Kimura, F., Satoh, A., Antioxidant effect of astaxanthin on phospholipid peroxidation in human erythrocytes (2011) Br J Nutr, 105, pp. 1563-1571Niki, E., Free radical initiators as source of water or lipid soluble peroxyl radicals (1990) Methods Enzymol, 186, pp. 100-108Niki, E., Action of ascorbic acid as a scavenger of active and stable oxygen radicals (1991) Am J Clin Nutr, 54, pp. 1119S-1124SOkamoto, K., Maruyama, T., Kaji, Y., Harada, M., Mawatari, S., Fujino, T., Verapamil prevents impairment in filterability of human erythrocytes exposed to oxidative stress (2004) Jpn J Physiol, 54, pp. 39-46Pandey, K.B., Rizvi, S.I., Biomarkers of oxidative stress in red blood cells (2011) Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 155, pp. 131-136Pandey, K.B., Mishra, N., Rizvi, S.I., Protein oxidation biomarkers in plasma of type 2 diabetic patients (2010) Clin Biochem, 43, pp. 508-511Pandey, K.B., Murtaza, M.M., Maurya, P.K., Rizvi, S.I., Plasma protein oxidation and its correlation with antioxidant potential during human aging (2010) Dis Markers, 29, pp. 31-36Rodrigues, E., Mariutti, L.R.B., Chisté, R.C., Mercadante, A.Z., Development of a novel micro-assay for evaluation of peroxyl radical scavenger capacity: Application to carotenoids and structure-activity relationship (2012) Food Chem, 135, pp. 2103-2111Sandhu, I.S., Ware, K., Grisham, M.B., Peroxyl radical-mediated hemolysis: Role of lipid, protein and sulfhydryl oxidation (1992) Free Radic Res Commun, 16, pp. 111-122Sato, Y., Kamo, S., Takahashi, T., Suzuki, Y., Mechanism of free radical-induced hemolysis of human erythrocytes: Hemolysis by water-soluble radical initiator (1995) Biochemistry, 34, pp. 8940-8949Silva, B.M., Santos, R.P., Mendes, L.S., Pinho, P.G., Valentão, P., Dracaena draco L. Fruit: Phytochemical and antioxidant activity assessment (2011) Food Res Int, 44, pp. 2182-2189Sundaram, S., Anjum, S., Dwivedi, P., Rai, G.K., Antioxidant activity and protective effect of banana peel against oxidative hemolysis of human erythrocyte at different stages of ripening (2011) Appl Biochem Biotechnol, 164, pp. 1192-1206Tedesco, I., Russo, M., Russo, P., Iacomino, G., Russo, G.L., Antioxidant effect of red wine polyphenols on red blood cells (2000) J Nutr Biochem, 11, pp. 114-119Woodall, A.A., Lee, S.W.-M., Weesie, R.J., Jackson, M.J., Britton, G., Oxidation of carotenoids by free radicals: Relationship between structure and reactivity (1997) Biochim Biophys Acta, 1336, pp. 33-42Woodall, A.A., Britton, J., Jackson, M., Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals: Relationship between carotenoids structure and protective ability (1997) Biochim Biophys Acta, 1336, pp. 575-586Wright, R.O., Lewander, W.J., Woolf, A.D., Methemoglobinemia: Etiology, pharmacology and clinical management (1999) Ann Emerg Med, 34, pp. 646-656Zou, C.-G., Agar, N.S., Jones, G.L., Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture (2001) Life Sci, 69, pp. 75-8

Efficiency Of Different Solvents On The Extraction Of Bioactive Compounds From The Amazonian Fruit Caryocar Villosum And The Effect On Its Antioxidant And Colour Properties

Caryocar villosum has been reported as a source of bioactive compounds that can be used as a potential product against oxidative damage in foods or biological systems. Objective - To obtain extracts from fruit pulps of C. villosum with high levels of bioactive compounds that have both antioxidant and colour properties. Method - The contents of bioactive compounds (total phenolic compounds, flavonoids, tannins, carotenoids and tocopherols), the colour parameters, the scavenging capacity against peroxyl radicals (ROO •) and the quenching activity against singlet oxygen ( 1O2) were determined. All data were used for extract classification by applying principal components analysis and hierarchical cluster analysis. Results - The water and ethanol:water (1:1, v/v) extracts presented the highest levels of total phenolic compounds (9.2 and 6.3 mg gallic acid equivalent/g extract, respectively), total flavonoids (3.8 and 2.5 mg catechin equivalent/g extract, respectively) and total tannins (7.6 and 2.4 mg tannic acid equivalent/g extract, respectively). The ethanol:water (1:1, v/v) extract also showed the highest scavenging capacity against ROO• (0.3 mmol trolox equivalent/g extract) and the highest protection against 1O2 (12.5%). On the other hand, the ethanol extracts, which were the most vivid and yellow colour (C*ab = 13.7 and b*= 13.3), presented the highest level of total carotenoids (0.1 mg/g), but low scavenging capacity against ROO• (0.01 mmol trolox equivalent/g extract). Conclusion - Based on these results and depending on the applicability, the ethanol:water, water and ethanol are the most promising solvents to obtain C. villosum extracts with high contents of bioactive compounds, ROO• scavenging capacity and protection against 1O2. Copyright © 2013 John Wiley & Sons, Ltd.254364372Almeida, M.R., Darin, J.D.C., Hernandes, L.C., Aissa, A.F., Chisté, R.C., Mercadante, A.Z., Antunes, L.M.G., Bianchi, M.L.P., Antigenotoxic effects of piquiá (Caryocar villosum) in multiple rat organs (2012) Plant Foods Hum Nutr, 67, pp. 171-177A.O.C.S official method ce 8-89 (reapproved 1997). Determination of tocopherols and tocotrienols in vegetable oils and fats by HPLC (1997) Official Methods and Recommended Practices of the American Oil Chemists' Society, p. 5. , AOCS. In, 5th edn. American Oil Chemists' Society: Champaign, ILBarreto, G.P.M., Benassi, M.T., Mercadante, A.Z., Bioactive compounds from several tropical fruits and correlation by multivariate analysis to free radical scavenger activity (2009) J Braz Chem Soc, 20, pp. 1856-1861Bligh, E.G., Dyer, W.J., A rapid method of total lipid extraction and purification (1959) Can J Biochem Physiol, 37, pp. 911-917Brune, M., Hallberg, L., Skånberg, A.B., Determination of iron-binding phenolic groups in foods (1991) J Food Sci, 56, pp. 128-131Chisté, R.C., Mercadante, A.Z., Identification and quantification, by HPLC-DAD-MS/MS, of carotenoids and phenolic compounds from the Amazonian fruit Caryocar villosum (2012) J Agric Food Chem, 60, pp. 5884-5892Chisté, R.C., Mercadante, A.Z., Gomes, A., Fernandes, E., Lima, J., Bragagnolo, N., In vitro scavenging capacity of annatto seed extracts against reactive oxygen and nitrogen species (2011) Food Chem, 127, pp. 419-426Chisté, R.C., Benassi, M.T., Mercadante, A.Z., Effect of solvent type on the extractability of bioactive compounds, antioxidant capacity and colour properties of natural annatto extracts (2011) Int J Food Sci Tech, 46, pp. 1863-1870Chisté, R.C., Freitas, M., Mercadante, A.Z., Fernandes, E., The potential of extracts of Caryocar villosum pulp to scavenge reactive oxygen and nitrogen species (2012) Food Chem, 135, pp. 1740-1749Clement, C.R., Piquiá (1993) Selected Species and Strategies to Enhance Income Generation from Amazonian Forests, pp. 108-114. , Food and Agriculture Organization: RomeLaying down specific purity criteria concerning colours for use in foodstuffs (1995) Off J Eur Comm, L226, pp. 1-41. , Commission Directive 95/45/ECDavies, B.H., (1976) Carotenoids: Chemistry and Biochemistry of Plant Pigments, pp. 38-165. , 2nd edn. Academic Press: LondonDe Rosso, V.V., Mercadante, A.Z., Identification and quantification of carotenoids, by HPLC-PDA-MS/MS, from Amazonian fruits (2007) J Agric Food Chem, 55, pp. 5062-5072De Rosso, V.V., Vieyra, F.E.M., Mercadante, A.Z., Borsarelli, C.D., Singlet oxygen quenching by anthocyanin's flavylium cations (2008) Free Radic Res, 42, pp. 885-891(1997) Guidance for Industry: Q3C Impurities: Residual Solvents, p. 16. , FDA. Food and Drug Administration, US Department of Health and Human Services: Rockville, MD, USAFinley, J.W., Kong, A.N., Hintz, K.J., Jeffery, E.H., Ji, L.L., Lei, X.G., Antioxidants in foods: State of the science important to the food industry (2011) J Agric Food Chem, 59, pp. 6837-6846Gomes, A., Fernandes, E., Silva, A.M.S., Santos, C.M.M., Pinto, D., Cavaleiro, J.A.S., Lima, J., 2-Styrylchromones: Novel strong scavengers of reactive oxygen and nitrogen species (2007) Bioorg Med Chem, 15, pp. 6027-6036Huang, D., Ou, B., Prior, R.L., The chemistry behind antioxidant capacity assays. J Agric (2005) Food Chem, 53, pp. 1841-1856International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (2005) Guidance for Industry, pp. 7-10. , ICH, Q2B Validation of Analytical Procedures: Methodology. International Conference on Harmonization Secretariat, c/o International Federation of Pharmaceutical Manufacturers and Associations: GenevaKimura, M., Rodriguez-Amaya, D.B., A scheme for obtaining standards and HPLC quantification of leafy vegetable carotenoids (2002) Food Chem, 78, pp. 389-398Kok, T., Waard, P., Wilms, L.C., Van Breda, S.G.J., Antioxidative and antigenotoxic properties of vegetables and dietary phytochemicals: The value of genomics biomarkers in molecular epidemiology (2010) Mol Nutr Food Res, 54, pp. 208-217Krinsky, N.I., The biological properties of carotenoids (1994) Pure Appl Chem, 66, pp. 1003-1010Marx, F., Andrade, E.H.A., Maia, J.G., Chemical composition of the fruit pulp of Caryocar villosum (1997) Eur Food Res Tech Z Lebensm-Unters Forsch A, 204, pp. 442-444Meléndez-Martínez, A.J., Britton, G., Vicario, I.M., Heredia, F.J., Relationship between the colour and the chemical structure of carotenoid pigments (2007) Food Chem, 101, pp. 1145-1150Montenegro, M.A., Rios, A.O., Mercadante, A.Z., Nazareno, M.A., Borsarelli, C.D., Model studies on the photosensitized isomerization of bixin (2004) J Agric Food Chem, 52, pp. 367-373Ou, B., Hampsch-Woodill, M., Prior, R.L., Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe (2001) J Agric Food Chem, 49, pp. 4619-4626Pajares, A., Bregliani, M., Montaña, M.P., Criado, S., Massad, W., Gianotti, J., Gutiérrez, I., García, N.A., Visible-light promoted photoprocesses on aqueous gallic acid in the presence of riboflavin. Kinetics and mechanism (2010) J Photochem Photobiol A-Chem, 209, pp. 89-94Pianovski, A.R., Vilela, A.F.G., Silva, A.A.S., Lima, C.G., Silva, K.K., Carvalho, V.F.M., Musis, C.R., Ferrari, M., Use of pequi oil (Caryocar brasiliense) in cosmetics emulsions: Development and evaluate of physical stability (2008) Braz J Pharm Sci, 44, pp. 249-259Singleton, V.L., Orthofer, R., Lamuela-Raventós Jr., R.M., Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent (1999) Meth Enz, 299, pp. 152-178Snyder, L.R., Carr, P.W., Rutan, S.C., Solvatochromically based solvent-selectivity triangle (1993) J Chromatogr A, 656, pp. 537-547Talcott, S.T., Krenek, K.A., Analysis methods of ellagitannins (2012) Analysis of Antioxidant-Rich Phytochemicals, pp. 181-205. , 1st edn, Xu Z. Howard L.R. (eds). Wiley-Blackwell: OxfordVatai, T., Škerget, M., Knez, Ž., Extraction of phenolic compounds from elderberry and different grape marc varieties using organic solvents and/or supercritical carbon dioxide (2009) J Food Eng, 90, pp. 246-254Waterman, P.G., Mole, S., (1994) Analysis of Phenolic Plant Metabolites, p. 238. , Blackwell Scientific Publications: OxfordYu, J., Ahmedna, M., Goktepe, I., Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics (2005) Food Chem, 90, pp. 199-206Zhishen, J., Mengcheng, T., Jianming, W., The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals (1999) Food Chem, 64, pp. 555-55

Fabrication of Through-Wafer Interconnections by Gold Electroplating

reserved5noA new method for conductive via’s using gold electroplating is presented. Tapered walls through wafer via (TWV) holes were made using a variable isotropy DRIE process, with a very good control over the obtained angles – angles of 11.3° and 21.8° were obtained with errors smaller than 10%. Barrier and seed layers were deposited in via’s performed by PVD (Physical Vapor Deposition) techniques with a very good coverage of the walls. Finally, gold electroplating was used to fill the narrow part of via’s.D. Vasilache; S. Colpo; F. Giacomozzi; B. Margesin; M. ChistèVasilache, Dan Adrian; Colpo, Sabrina; Giacomozzi, Flavio; Margesin, Benno; Chistè, Matte

Carotenoids Are Effective Inhibitors Of In Vitro Hemolysis Of Human Erythrocytes, As Determined By A Practical And Optimized Cellular Antioxidant Assay

β-Carotene, zeaxanthin, lutein, β-cryptoxanthin, and lycopene are liposoluble pigments widely distributed in vegetables and fruits and, after ingestion, these compounds are usually detected in human blood plasma. In this study, we evaluated their potential to inhibit hemolysis of human erythrocytes, as mediated by the toxicity of peroxyl radicals (ROO•). Thus, 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) was used as ROO• generator and the hemolysis assay was carried out in experimental conditions optimized by response surface methodology, and successfully adapted to microplate assay. The optimized conditions were verified at 30 × 106 cells/mL, 17 mM of AAPH for 3 h, at which 48 ± 5% of hemolysis was achieved in freshly isolated erythrocytes. Among the tested carotenoids, lycopene (IC50 = 0.24 ± 0.05 μM) was the most efficient to prevent the hemolysis, followed by β-carotene (0.32 ± 0.02 μM), lutein (0.38 ± 0.02 μM), and zeaxanthin (0.43 ± 0.02 μM). These carotenoids were at least 5 times more effective than quercetin, trolox, and ascorbic acid (positive controls). β-Cryptoxanthin did not present any erythroprotective effect, but rather induced a hemolytic effect at the highest tested concentration (3 μM). These results suggest that selected carotenoids may have potential to act as important erythroprotective agents by preventing ROO•-induced toxicity in human erythrocytes. Practical Application: Selected carotenoids may have potential to be used in the development of phytopharmaceutical products as important erythroprotective agents by preventing ROO•-induced toxicity in human erythrocytes.799H1841H1847Banerjee, A., Kunwar, A., Mishra, B., Priyadarsini, K.I., Concentration dependent antioxidant/pro-oxidant activity of curcumin Studies from AAPH induced hemolysis of RBCs (2008) Chem-Biol Interact, 174 (2), pp. 134-139Chisté, R.C., Mercadante, A.Z., Gomes, A., Fernandes, E., Lima, J.L.F.D., Bragagnolo, N., In vitro scavenging capacity of annatto seed extracts against reactive oxygen and nitrogen species (2011) Food Chem, 127 (2), pp. 419-426Chisté, R.C., Freitas, M., Mercadante, A.Z., Fernandes, E., The potential of extracts of Caryocar villosum pulp to scavenge reactive oxygen and nitrogen species (2012) Food Chem, 135 (3), pp. 1740-1749Chisté, R.C., Freitas, M., Mercadante, A.Z., Fernandes, E., Carotenoids inhibit lipid peroxidation and hemoglobin oxidation, but not the depletion of glutathione induced by ROS in human erythrocytes (2014) Life Sci, 99 (1-2), pp. 52-60Di Mascio, P., Kaiser, S., Sies, H., Lycopene as the most efficient biological carotenoid singlet oxygen quencher (1989) Arch Biochem Biophys, 274 (2), pp. 532-538El-Agamey, A., Lowe, G.M., McGarvey, D.J., Mortensen, A., Phillip, D.M., Truscott, T.G., Young, A.J., Carotenoid radical chemistry and antioxidant/pro-oxidant properties (2004) Arch Biochem Biophys, 430 (1), pp. 37-48Guo, J.J., Hu, C.H., Mechanism of chain termination in lipid peroxidation by carotenes: a theoretical study (2010) J Phys Chem B, 114 (50), pp. 16948-16958Jomova, K., Kysel, O., Madden, J.C., Morris, H., Enoch, S.J., Budzak, S., Young, A.J., Valko, M., Electron transfer from all-trans- β-carotene to the t-butyl peroxyl radical at low oxygen pressure (an EPR spectroscopy and computational study) (2009) Chem Phys Lett, 478 (4-6), pp. 266-270Kiko, T., Nakagawa, K., Tsuduki, T., Suzuki, T., Arai, H., Miyazawa, T., Significance of lutein in red blood cells of Alzheimer's disease patients (2012) J Alzheimers Dis, 28 (3), pp. 593-600Ko, F.N., Hsiao, G., Kuo, Y.H., Protection of oxidative hemolysis by demethyldiisoeugenol in normal and β-thalassemic red blood cells (1997) Free Radical Bio Med, 22 (1-2), pp. 215-222Kok, T.M.C.M., de Waard, P., Wilms, L.C., van Breda, S.G.J., Antioxidative and antigenotoxic properties of vegetables and dietary phytochemicals: the value of genomics biomarkers in molecular epidemiology (2010) Mol Nutr Food Res, 54 (2), pp. 208-217Lian, F.Z., Hu, K.Q., Russell, R.M., Wang, X.D., β-Cryptoxanthin suppresses the growth of immortalized human bronchial epithelial cells and non-small-cell lung cancer cells and up-regulates retinoic acid receptor β expression (2006) Int J Cancer, 119 (9), pp. 2084-2089Lorenzo, Y., Azqueta, A., Luna, L., Bonilla, F., Dominguez, G., Collins, A.R., The carotenoid b-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells (2009) Carcinogenesis, 30 (2), pp. 308-314Magalhães, A.S., Silva, B.M., Pereira, J.A., Andrade, P.B., Valentao, P., Carvalho, M., Protective effect of quince (Cydonia oblonga Miller) fruit against oxidative hemolysis of human erythrocytes (2009) Food Chem Toxicol, 47 (6), pp. 1372-1377Miyazawa, T., Suzuki, T., Yasuda, K., Fujimoto, K., Meguro, K., Sasaki, H., Accumulation of phospholipid hydroperoxides in red blood cell membranes in Alzheimer disease (1992) Oxygen Radicals, pp. 327-330. , Yagi, K., Kondo, M., Niki, E., Yoshikawa, T., editors Amsterdam: Elsevier Science PublishingMüller, L., Theile, K., Böhm, V., In vitro antioxidant activity of tocopherols and tocotrienols and comparison of vitamin E concentration and lipophilic antioxidant capacity in human plasma (2010) Mol Nutr Food Res, 54 (5), pp. 731-742Naguib, Y.M.A., Antioxidant activities of astaxanthin and related carotenoids (2000) J Agric Food Chem, 48 (4), pp. 1150-1154Nakagawa, K., Kiko, T., Hatade, K., Asai, A., Kimura, F., Sookwong, P., Tsuduki, T., Miyazawa, T., Development of a high-performance liquid chromatography-based assay for carotenoids in human red blood cells: application to clinical studies (2008) Anal Biochem, 381 (1), pp. 129-134Niki, E., Free-radical initiators as source of water-soluble or lipid-soluble peroxyl radicals (1990) Method Enzymol, 186, pp. 100-108Niki, E., Action of ascorbic-acid as a scavenger of active and stable oxygen radicals (1991) Am J Clin Nutr, 54 (6), pp. S1119-S1124Ou, B.X., Hampsch-Woodill, M., Prior, R.L., Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe (2001) J Agric Food Chem, 49 (10), pp. 4619-4626Rodrigues, E., Mariutti, L.R.B., Chiste, R.C., Mercadante, A.Z., Development of a novel micro-assay for evaluation of peroxyl radical scavenger capacity: Application to carotenoids and structure-activity relationship (2012) Food Chem, 135 (3), pp. 2103-2111Sato, Y., Kamo, S., Takahashi, T., Suzuki, Y., Mechanism of free radical-induced hemolysis of human erythrocytes-hemolysis by water-soluble radical initiator (1995) Biochemistry, 34 (28), pp. 8940-8949Silva, B.M., Santos, R.P., Mendes, L.S., de Pinho, P.G., Valentao, P., Andrade, P.B., Pereira, J.A., Carvalho, M., Dracaena draco L. fruit: phytochemical and antioxidant activity assessment (2011) Food Res Int, 44 (7), pp. 2182-2189Sundaram, S., Anjum, S., Dwivedi, P., Rai, G.K., Antioxidant activity and protective effect of banana peel against oxidative hemolysis of human erythrocyte at different stages of ripening (2011) Appl Biochem Biotech, 164 (7), pp. 1192-1206Zou, C.G., Agar, N.S., Jones, G.L., Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture (2001) Life Sci, 69 (1), pp. 75-8

A multi-analytical approach to the use of conifer needles as passive samplers of particulate matter and organic pollutants

The present work regards the analysis of airborne pollutants emitted by an electric arc furnace steel making plant at a test site in Northern Italy and collected by conifer needles. The spatial and temporal trends of accumulation of Mn, Zn, Fe, Cr, Pb, polychlorinated dibenzo-p-dioxins/dibenzofurans and polychlorinated biphenyls dioxin revealed that the contribution from the steel making plant has never been particularly high, whereas traffic emerged as a significant pollution source. The benefits of combining bulk and single particle analysis in air pollution studies from different sources are also discussed. \ua9 Taiwan Association for Aerosol Research

PCDD/Fs in the soils in the province of Trento: 10\ua0years of monitoring

This paper presents a 10-year overview of the dioxins and furans (PCDD/Fs) content in soils in the province of Trento (Italy). The aim was to compare the results found in the Valsugana valley where there is a steel-making plant with other locations within the province. During 2002 and from 2005 to 2010, campaigns were carried out in order to obtain a background reference in terms of micropollutants, in view of the possible construction of a municipal solid waste (MSW) incinerator in Trento. In 2009, a campaign was performed for the environmental characterization of the Valsugana valley, the town of Trento and its surroundings, in order to help assess the impact of the steel-making plant. In 2012, another campaign was carried out by the Department of Civil and Environmental Engineering of Trento, in order to monitor the soils in the area around the steel mill. All the campaigns showed relatively low concentrations of PCDD/Fs, both in protected areas and in the areas close to the industrial plants. No critical situations were identified, as also confirmed by an estimation of the potential daily PCDD/F intake by children subject to accidental ingestion of soil